计算溶液所需的质量、体积或浓度。
组织/细胞/细菌总RNA提取试剂盒 (DNase I, 含保存液)
有货
为什么选择此级别
BioReagent,分子生物学级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
2-8°C储存,室温。低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
本试剂盒提供了从RNA保存到提取的全套解决方案。试剂盒中的组织样本RNA保存液是 一种液态的、无毒的组织样本RNA保存液，它能迅速渗入组织，通过高效抑制RNase活性从而保护非冷冻细胞RNA于原位，使其更适合于组织基因表达谱的分析。该试剂可广泛用于新鲜动物组织样本如肝脏、脑、肺、脾脏、肾脏、心脏、肌肉等以及某些植物样本的保存，特别适合野外采集的样本、大批量采集的动物组织或不能立即进行冻存的样本。加入组织样本 RNA 保存液的样品可在37℃稳定保存1天，25℃稳定保存1周，4℃稳定保存1个月，-20℃或-80℃条件下长期保存。经该产品保护的组织样品可用于所有关于RNA的后续实验，包括总RNA的提取、micro RNA的提取和mRNA的提取等。
对于RNA提取，试剂盒采用了独特的裂解系统，适合从动物组织、细胞和细菌样本中提取总RNA。使用本试剂盒可以有效提取分子量大于200 nt的RNA。本试剂盒具有高效、快速、方便的特点，提取过程中也无需苯酚氯仿抽提等步骤。利用该试剂盒提取的RNA纯度高，极少含蛋白质、基因组DNA和其他杂质的污染。提取得到的RNA可以直接用于Northern杂交、斑点杂交、mRNA纯化、体外翻译、RNA分解酶的保护分析、RT-PCR、Real Time RT-PCR和构建cDNA文库等各种分子生物学实验。
注意事项
1. 操作前在Buffer TRL 中加入β-巯基乙醇至终浓度1%，配好的TRL 可在4℃放置1个月。Buffer TRL 在储存时可能会形成沉淀，如果有沉淀出现，请于37℃加热溶解，然后待恢复至室温后使用。
2. 尽可能使用新鲜收集的样品，RNA产量与起始样品中RNA的完整性有关，按照说明书标准流程降解为小片段的RNA不能被有效回收。
3. DNase I 储存液的配制：用1 mL注射器将550 μL RNase-Free ddH₂O注入装有DNase I 干粉的瓶中，轻柔混匀至溶解，分装后-30~-15℃储存（可保存9个月），每次根据用量需求取出。配制的DNase I 工作液置于2-8℃储存（可保存42天），避免反复冻融。
4. 如需提取包含Small RNA (< 200 nt) 的RNA，可以联系获得相应的提取流程。
样本保存步骤
一、保存新鲜组织样本
1. 样本处理
1.1 动物组织
将组织剪切成合适大小后（建议组织块长宽高均不大于0.5 cm），然后迅速完全浸泡于5 - 10倍体积的组织样本RNA保存液中，一般1 g组织需5 mL保存液。
注意：已浸入组织样本RNA保存液中组织样品放置一段时间后，RNase酶已失活，可从保存液中取出并剪切成更小的组织块，然后重新放回保存液中。本试剂不适合保存液体渗透性弱的组织如骨头等样本。
1.2 植物组织
本试剂对于柔软的植物组织（如嫩叶、嫩茎）能直接渗透，起到理想的RNA保护作用。将组织剪切成合适大小后（建议组织块长宽高均不大于0.5cm），然后迅速完全浸泡于5 - 10倍体积的组织样本RNA保存液中。
注意：若用本产品保存植物叶片组织，则需将叶片表面蜡表皮破坏，否则保存液很难完全渗入组织中。
2. 样品保存
加入保存液的样品可在37℃稳定保存1天，25℃稳定保存1周，4℃稳定保存1个月，-20℃或-80℃条件下长期保存。
3. RNA提取
取出保存的组织样品，立即开始RNA提取或进行其他处理。
二、保存培养细胞，悬浮细胞和细菌
1. 标记收集管。
2. 将待保存的细胞样品转移到离心管中，离心收集细胞/细菌，弃上清。
3. 用冰浴的缓冲液（PBS）洗涤一次。
4. 将细胞悬浮在少量缓冲液中。
5. 加入5 - 10倍体积的组织样本RNA保存液，混匀。
6. 加入保存液的样品可在37℃稳定保存1天，25℃稳定保存1周，4℃稳定保存1个月，-20℃ 或-80℃条件下长期保存。
7. RNA提取前样品处理：
7.1 4℃保存的细胞样品需先离心，倒弃保护液并收集细胞。
7.2 -20℃或-80℃保存的样品需先在室温下融化，然后离心，倒弃保护液并收集细胞。
8. 立即开始RNA提取或进行其他处理。
三、保存全血中的白细胞
1. 样本处理
先将白细胞从全血中分离出来。
注意：不要将全血、血浆或血清直接保存于组织样本RNA保存液，因为它们蛋白含量过高，易与保存液混合后形成沉淀。
2. 用冰浴的缓冲液（PBS）洗涤一次，并将白细胞悬浮在少量缓冲液（PBS）中。
3. 加入5 - 10倍体积的组织样本RNA保存液，混匀。
4. 加入保存液的样品可在37℃稳定保存1天，25℃稳定保存1周，4℃稳定保存1个月，-20℃ 或-80℃条件下长期保存。
5. 立即开始RNA提取或进行其他处理。
操作步骤
使用前准备：在12 mL Buffer RW2 中加入48 mL的无水乙醇。
一、从组织中提取总RNA
1. 匀浆处理
每 10–20 mg 组织样本（RNA保存液保存的组织或新鲜的组织）加入 300 μL Buffer TRL （使用前请先加入 β-巯基乙醇），用研磨杵将组织彻底研磨（如组织较难彻底研磨，可选用电动或玻璃匀浆器）。随后向组织匀浆液中加入 590 μL RNase-Free ddH₂O 和 10 μL Proteinase K，混匀后 56℃ 孵育 10–20 min。
2. 14,000 rpm（18,630 × g）离心 2 min，将上清转移至另一离心管中。
3. 缓慢加入 0.5 倍滤液体积 的无水乙醇，充分混匀，此时可能会出现沉淀，将得到的溶液和沉淀一起转入吸附柱 RA（吸附柱放在收集管中），每次上样量 ≤700 μL（如果混合液体积超过 700 μL，需要分次转入），12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
4. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
5. DNase I 工作液的配制：取 10 μL DNase I 储存液放入新的 RNase-Free 离心管中，加入 70 μL DNase I Buffer，轻柔混匀。
6. 向吸附柱中央加入 80 μL 的 DNase I 工作液，室温放置 15 min。
7. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
8. 向吸附柱中加入 550 μL Buffer RW2（使用前请先加入无水乙醇），12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
9. 重复步骤 8。
10. 12,000 rpm（13,680 × g）离心 2 min。
11. 将吸附柱放入一个新的 1.5 mL RNase-Free 离心管中，开盖晾干 2 min，以彻底挥发乙醇。向吸附膜中央加入 30–100 μL RNase-Free ddH₂O，室温放置 1 min，12,000 rpm（13,680 × g）离心 30 sec，得到 RNA 溶液。
二、从培养细胞中提取总RNA
1. 匀浆处理
1.1 不使用组织样本RNA保存液时：
- 贴壁细胞：彻底吸弃培养液，每 6–10 cm² 面积加入 600 μL Buffer TRL （使用前请先加入 β-巯基乙醇），用移液器吹打 3–5 次使细胞裂解。
- 细胞悬液：500 × g 离心收集细胞，每 5×10⁶–1×10⁷ 细胞加入 600 μl Buffer TRL （使用前请加入 β-巯基乙醇），少于 5×10⁶ 细胞加入 350 μL Buffer TRL（使用前请先加入 β-巯基乙醇），用移液器吹打 3–5 次使细胞裂解。
1.2 使用组织样本RNA保存液时：
收集保存于组织样本RNA保存液中的细胞，每 1×10⁶–1×10⁷ 细胞加入 600 μL Buffer TRL（使用前请先加入 β-巯基乙醇），少于 5×10⁶ 细胞加入 350 μL Buffer TRL（使用前请先加入 β-巯基乙醇），用移液器吹打 3–5 次使细胞裂解。
2. （可选） 将所有溶液转移至过滤柱 CS（客户自备，过滤柱放在收集管中），12,000 rpm（13,680 × g）离心 2 min，小心吸取收集管中的滤液至新的 1.5 mL RNase-Free 离心管中，吸头尽量避免接触收集管中的细胞碎片沉淀。
3. 缓慢加入 0.5 倍滤液体积 的无水乙醇，充分混匀，此时可能会出现沉淀，将得到的溶液和沉淀一起转入吸附柱 RA（吸附柱放在收集管中），12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
4. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
5. DNase I 工作液的配制：取 10 μL DNase I 储存液放入新的 RNase-Free 离心管中，加入 70 μL DNase I Buffer，轻柔混匀。
6. 向吸附柱中央加入 80 μL 的 DNase I 工作液，室温放置 15 min。
7. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，吸附柱放回收集管中。
8. 向吸附柱中加入 550 μL Buffer RW2（使用前请先加入无水乙醇），12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
9. 重复步骤 8。
10. 12,000 rpm（13,680 × g）离心 2 min。
11. 将吸附柱放入一个新的 1.5 mL RNase-Free 离心管中，开盖晾干 2 min，以彻底挥发乙醇。向吸附膜中央加入 30–100 μL RNase-Free ddH₂O，室温放置 1 min，12,000 rpm（13,680 × g）离心 30 sec，得到 RNA 溶液。
三、从细菌中提取总RNA
1. 按照本说明书组织样本RNA保存液使用方法收集菌体（菌体量请不要超过 1×10⁹），小心去除所有上清。
2. 用含有溶菌酶的 100 μL TE 缓冲液彻底重悬菌体（客户自配），37℃ 孵育。溶菌酶使用量及孵育时间见下表。
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TE缓冲液配方：50 mM Tris，pH 8.0；10 mM Na₂-EDTA，pH 8.0。121℃ 灭菌处理 20 min。
例如：
G⁻ 细菌：1 μL 溶菌酶溶液（50 mg/mL）+ 99 μL TE缓冲液，重悬菌体，溶菌酶终浓度即为 500 μg/mL；
G⁺ 细菌：6 μL 溶菌酶溶液（50 mg/mL）+ 94 μL TE缓冲液，重悬菌体，溶菌酶终浓度即为 3 mg/mL。
3. 加入 350 μL Buffer TRL（使用前请先加入 β-巯基乙醇），涡旋振荡混匀（此步骤可能出现沉淀），若出现不溶性沉淀，12,000 rpm（13,680 × g）离心 2 min，将上清转移至另一离心管中。
4. 缓慢加入 0.5 倍滤液体积 的无水乙醇，充分混匀，此时可能会出现沉淀，将得到的溶液和沉淀一起转入吸附柱 RA（吸附柱放在收集管中），12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
5. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
6. DNase I 工作液的配制：取 10 μL DNase I 储存液放入新的 RNase-Free 离心管中，加入 70 μL DNase I Buffer，轻柔混匀。
7. 向吸附柱中央加入 80 μL 的 DNase I 工作液，室温放置 15 min。
8. 向吸附柱中加入 350 μL Buffer RW，12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
9. 向吸附柱中加入 550 μL Buffer RW2（使用前请先加入无水乙醇），12,000 rpm（13,680 × g）离心 30 sec，弃废液，将吸附柱放回收集管中。
10. 重复步骤 9。
11. 12,000 rpm（13,680 × g）离心 2 min。
12. 将吸附柱放入一个新的 1.5 mL RNase-Free 离心管中，开盖晾干 2 min，以彻底挥发乙醇。向吸附膜中央加入 30–100 μL RNase-Free ddH₂O，室温放置 1 min，12,000 rpm（13,680 × g）离心 30 sec，得到 RNA 溶液。
RNA 完整性、浓度及纯度检测
完整性：
RNA 可用普通琼脂糖凝胶电泳（电泳条件：胶浓度 1–1.2%；0.5× TBE 电泳缓冲液；120–150 V，15–25 min）检测完整性。由于 rRNA 占 RNA 总量的 80% 以上，电泳后 UV 下应能看到非常明显的 rRNA 条带。不同物种 rRNA 类型及大小会有差异：
- 动物：28S（4.7–5.0 kb）、18S（1.9 kb）、5S（120 bp）
- 植物：25S（3.7 kb）、18S（1.9 kb）、5S（120 bp）
- 大肠杆菌：23S（2.9 kb）、16S（1.5 kb）、5S（120 bp）
- 昆虫/软体动物：18S 通常会比 28S 条带亮。
条带模糊且弥散拖尾一般表明 RNA 严重降解，建议重新电泳或提取，否则 qPCR 可能出 Ct 值，但准确性与重复性不佳。
浓度：
通过测量分光光度计中 260 nm 处的吸光度（A₂₆₀）确定 RNA 浓度，用 RNase-Free ddH₂O 进行调零，260 nm 处 1 个单位的吸光度相当于 40 μg/mL RNA。
纯度：
A₂₆₀/A₂₈₀ 比值可以用来评估核酸纯度。A₂₆₀/A₂₈₀ 比值在 1.8–2.1 之间时，意味着高纯度的 RNA。A₂₆₀/A₂₈₀ 比值同时依赖于 pH 值及离子强度；当 pH 升高时，A₂₈₀ 读数会降低，而 A₂₆₀ 则不受影响，这会导致 A₂₆₀/A₂₈₀ 比值的升高。由于水通常都具有酸性 pH 值，它会降低 A₂₆₀/A₂₈₀ 比值。我们推荐使用弱碱性 pH 的缓冲溶液（例如 TE，pH 8.0）作为稀释液及空白对照，以确保得到精确、可重复的读数。
This kit provides a complete solution from RNA preservation to extraction. The Tissue Sample RNA Preservation Solution is a liquid, non‑toxic RNA preservation solution for tissue samples. It rapidly penetrates tissues and protects RNA in non‑frozen cells in situ by efficiently inhibiting RNase activity, making it more suitable for gene expression profiling of tissues. This solution can be widely used for the preservation of fresh animal tissue samples such as liver, brain, lung, spleen, kidney, heart, muscle, and some plant samples. It is especially suitable for samples collected in the field, large batches of animal tissues, or samples that cannot be frozen immediately. Samples preserved in the Tissue Sample RNA Preservation Solution can be stored stably for 1 day at 37°C, 1 week at 25°C, 1 month at 4°C, and for long‑term storage at -20°C or -80°C. Tissue samples preserved with this product can be used in all downstream RNA experiments, including total RNA extraction, microRNA extraction, and mRNA extraction.
For RNA extraction, the kit employs a unique lysis system suitable for total RNA extraction from animal tissues, cells, and bacterial samples. Using this kit, RNA larger than 200 nt can be efficiently extracted. The kit is efficient, rapid, and convenient, and the extraction process does not require phenol‑chloroform extraction steps. The RNA purified with this kit is of high purity, with minimal contamination from proteins, genomic DNA, and other impurities. The extracted RNA can be directly used in various molecular biology experiments, including Northern blot, dot blot, mRNA purification, in vitro translation, RNase protection assay, RT‑PCR, real‑time RT‑PCR, and cDNA library construction.
Precautions
Before use, add β‑mercaptoethanol to Buffer TRL to a final concentration of 1%. The prepared TRL can be stored at 4°C for 1 month. If precipitate forms in Buffer TRL, heat at 37°C to dissolve, then allow to return to room temperature before use.
Use freshly collected samples whenever possible. RNA yield depends on the integrity of the RNA in the starting material. RNA degraded into small fragments following the standard protocol cannot be efficiently recovered.
Preparation of DNase I stock solution: Use a 1 mL syringe to inject 550 μL RNase‑Free ddH₂O into the vial containing DNase I powder. Mix gently until dissolved. Aliquot and store at -30 to -15°C (can be stored for 9 months). Thaw aliquots as needed. The prepared DNase I working solution can be stored at 2‑8°C for up to 42 days. Avoid repeated freeze‑thaw cycles.
If extraction of RNA containing small RNA (<200 nt) is required, please contact us for the corresponding protocol.
Sample Preservation Procedure
Preserving Fresh Tissue Samples
1. Sample Processing
1.1 Animal tissue
Cut the tissue into appropriate sizes (recommended dimensions ≤0.5 cm in each direction) and quickly immerse completely in 5–10 volumes of Tissue Sample RNA Preservation Solution (generally 5 mL of solution per 1 g of tissue).
Note: After tissue has been immersed in the preservation solution for some time, RNase is inactivated. The tissue can be removed, cut into smaller pieces, and returned to the solution. This solution is not suitable for preserving tissues with low liquid permeability, such as bone.
1.2 Plant tissue
This solution can directly penetrate soft plant tissues (e.g., young leaves, young stems) and provides ideal RNA protection. Cut the tissue into appropriate sizes (recommended dimensions ≤0.5 cm in each direction) and quickly immerse completely in 5–10 volumes of Tissue Sample RNA Preservation Solution.
Note: When preserving plant leaf tissue, the waxy cuticle on the leaf surface must be disrupted; otherwise, the solution will not fully penetrate the tissue.
2. Sample Storage
Samples in the preservation solution can be stored stably for 1 day at 37°C, 1 week at 25°C, 1 month at 4°C, and for long‑term storage at -20°C or -80°C.
3. RNA Extraction
Remove the preserved tissue sample and immediately proceed with RNA extraction or other processing.
Preserving Cultured Cells, Suspension Cells, and Bacteria
1. Label collection tubes.
2. Transfer the cells/bacteria to be preserved to a centrifuge tube, pellet by centrifugation, and discard the supernatant.
3. Wash once with ice‑cold buffer (e.g., PBS).
4. Resuspend the cells in a small volume of buffer.
5. Add 5–10 volumes of Tissue Sample RNA Preservation Solution and mix well.
6. Samples in the preservation solution can be stored stably for 1 day at 37°C, 1 week at 25°C, 1 month at 4°C, and for long‑term storage at -20°C or -80°C.
7. Sample processing before RNA extraction:
7.1 For cells stored at 4°C: centrifuge, discard the preservation solution, and collect the cell pellet.
7.2 For cells stored at -20°C or -80°C: thaw at room temperature, then centrifuge, discard the preservation solution, and collect the cell pellet.
8. Immediately proceed with RNA extraction or other processing.
Preserving White Blood Cells from Whole Blood
1. Sample processing
First, isolate white blood cells from whole blood.
Note: Do not preserve whole blood, plasma, or serum directly in the Tissue Sample RNA Preservation Solution, as their high protein content may form a precipitate when mixed with the solution.
2. Wash once with ice‑cold buffer (PBS) and resuspend the white blood cells in a small volume of buffer (PBS).
3. Add 5–10 volumes of Tissue Sample RNA Preservation Solution and mix well.
4. Samples in the preservation solution can be stored stably for 1 day at 37°C, 1 week at 25°C, 1 month at 4°C, and for long‑term storage at -20°C or -80°C.
5. Immediately proceed with RNA extraction or other processing.
Protocol
Preparation before use: Add 48 mL of absolute ethanol to 12 mL of Buffer RW2.
Total RNA Extraction from Tissues
1. Homogenization
For every 10–20 mg of tissue (preserved in RNA preservation solution or fresh), add 300 μL Buffer TRL(add β‑mercaptoethanol before use). Grind the tissue thoroughly with a pestle (if the tissue is difficult to grind, use an electric or glass homogenizer). Then add 590 μL RNase‑Free ddH₂O and 10 μL Proteinase K to the homogenate. Mix and incubate at 56°C for 10–20 minutes.
2. Centrifuge at 14,000 rpm (18,630 × g) for 2 minutes. Transfer the supernatant to a new centrifuge tube.
3. Slowly add 0.5 volumes of absolute ethanol (relative to the supernatant volume) and mix thoroughly. A precipitate may appear. Transfer the entire solution and precipitate to a Spin Column RA (placed in a collection tube). Load no more than 700 μL per spin (if the mixture volume exceeds 700 μL, load in multiple spins). Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
4. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
5. Preparation of DNase I working solution: Transfer 10 μL of DNase I stock solution to a new RNase‑Free centrifuge tube, add 70 μL DNase I Buffer, and mix gently.
6. Add 80 μL of the DNase I working solution to the center of the column. Let stand at room temperature for 15 minutes.
7. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
8. Add 550 μL Buffer RW2 (ensure absolute ethanol has been added before use) to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
9. Repeat step 8.
10. Centrifuge at 12,000 rpm (13,680 × g) for 2 minutes.
11. Transfer the column to a new 1.5 mL RNase‑Free centrifuge tube. Open the lid and air‑dry for 2 minutes to completely evaporate any residual ethanol. Add 30–100 μL RNase‑Free ddH₂O to the center of the membrane. Let stand at room temperature for 1 minute, then centrifuge at 12,000 rpm (13,680 × g) for 30 seconds to collect the RNA solution.
Total RNA Extraction from Cultured Cells
1. Homogenization
1.1 Without using Tissue Sample RNA Preservation Solution:
Adherent cells: Completely aspirate the culture medium. For every 6–10 cm² of growth area, add 600 μL Buffer TRL(add β‑mercaptoethanol before use). Pipette 3–5 times to lyse the cells.
Cell suspension: Pellet cells by centrifugation at 500 × g. For 5×10⁶–1×10⁷ cells, add 600 μL Buffer TRL(add β‑mercaptoethanol before use); for fewer than 5×10⁶ cells, add 350 μL Buffer TRL. Pipette 3–5 times to lyse the cells.
1.2 Using Tissue Sample RNA Preservation Solution:
Collect cells preserved in the Tissue Sample RNA Preservation Solution. For 1×10⁶–1×10⁷ cells, add 600 μL Buffer TRL(add β‑mercaptoethanol before use); for fewer than 5×10⁶ cells, add 350 μL Buffer TRL. Pipette 3–5 times to lyse the cells.
2. (Optional) Transfer the entire solution to a Filter Column CS (user‑supplied, placed in a collection tube). Centrifuge at 12,000 rpm (13,680 × g) for 2 minutes. Carefully transfer the flow‑through to a new 1.5 mL RNase‑Free centrifuge tube, avoiding the cell debris pellet.
3. Slowly add 0.5 volumes of absolute ethanol (relative to the flow‑through volume) and mix thoroughly. A precipitate may appear. Transfer the entire solution and precipitate to a Spin Column RA (placed in a collection tube). Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
4. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
5. Preparation of DNase I working solution: Transfer 10 μL of DNase I stock solution to a new RNase‑Free centrifuge tube, add 70 μL DNase I Buffer, and mix gently.
6. Add 80 μL of the DNase I working solution to the center of the column. Let stand at room temperature for 15 minutes.
7. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
8. Add 550 μL Buffer RW2 (ensure absolute ethanol has been added before use) to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
9. Repeat step 8.
10. Centrifuge at 12,000 rpm (13,680 × g) for 2 minutes.
11. Transfer the column to a new 1.5 mL RNase‑Free centrifuge tube. Open the lid and air‑dry for 2 minutes to completely evaporate any residual ethanol. Add 30–100 μL RNase‑Free ddH₂O to the center of the membrane. Let stand at room temperature for 1 minute, then centrifuge at 12,000 rpm (13,680 × g) for 30 seconds to collect the RNA solution.
Total RNA Extraction from Bacteria
1. Collect bacterial cells according to the Tissue Sample RNA Preservation Solution instructions (do not exceed 1×10⁹ cells). Carefully remove all supernatant.
2. Thoroughly resuspend the cells in 100 μL of TE buffer containing lysozyme (user‑prepared). Incubate at 37°C. The amount of lysozyme and incubation time are shown in the table below:
|
TE buffer composition: 50 mM Tris, pH 8.0; 10 mM Na₂‑EDTA, pH 8.0. Sterilize by autoclaving at 121°C for 20 minutes.
Example:
G⁻ bacteria: 1 μL lysozyme solution (50 mg/mL) + 99 μL TE buffer to resuspend the cells → final lysozyme concentration 500 μg/mL.
G⁺ bacteria: 6 μL lysozyme solution (50 mg/mL) + 94 μL TE buffer to resuspend the cells → final lysozyme concentration 3 mg/mL.
3. Add 350 μL Buffer TRL(add β‑mercaptoethanol before use). Vortex to mix (a precipitate may appear). If insoluble precipitate remains, centrifuge at 12,000 rpm (13,680 × g) for 2 minutes and transfer the supernatant to a new centrifuge tube.
4. Slowly add 0.5 volumes of absolute ethanol (relative to the supernatant volume) and mix thoroughly. A precipitate may appear. Transfer the entire solution and precipitate to a Spin Column RA (placed in a collection tube). Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
5. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
6. Preparation of DNase I working solution: Transfer 10 μL of DNase I stock solution to a new RNase‑Free centrifuge tube, add 70 μL DNase I Buffer, and mix gently.
7. Add 80 μL of the DNase I working solution to the center of the column. Let stand at room temperature for 15 minutes.
8. Add 350 μL Buffer RW to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
9. Add 550 μL Buffer RW2 (ensure absolute ethanol has been added before use) to the column. Centrifuge at 12,000 rpm (13,680 × g) for 30 seconds. Discard the flow‑through and return the column to the collection tube.
10. Repeat step 9.
11. Centrifuge at 12,000 rpm (13,680 × g) for 2 minutes.
12. Transfer the column to a new 1.5 mL RNase‑Free centrifuge tube. Open the lid and air‑dry for 2 minutes to completely evaporate any residual ethanol. Add 30–100 μL RNase‑Free ddH₂O to the center of the membrane. Let stand at room temperature for 1 minute, then centrifuge at 12,000 rpm (13,680 × g) for 30 seconds to collect the RNA solution.
RNA Integrity, Concentration, and Purity Assessment
Integrity:
RNA integrity can be assessed by standard agarose gel electrophoresis (conditions: 1–1.2% gel, 0.5× TBE running buffer, 120–150 V for 15–25 minutes). Since rRNA accounts for more than 80% of total RNA, very distinct rRNA bands should be visible under UV after electrophoresis. The types and sizes of rRNA vary among species:
Animals: 28S (4.7–5.0 kb), 18S (1.9 kb), 5S (120 bp)
Plants: 25S (3.7 kb), 18S (1.9 kb), 5S (120 bp)
E. coli: 23S (2.9 kb), 16S (1.5 kb), 5S (120 bp)
Insects/Mollusks: The 18S band is typically brighter than the 28S band.
If the bands are smeared and diffuse, it generally indicates severe RNA degradation. Re‑run the gel or re‑extract the RNA. Otherwise, qPCR may still yield Ct values, but accuracy and reproducibility will be poor.
Concentration:
Determine RNA concentration by measuring absorbance at 260 nm (A₂₆₀) using a spectrophotometer. Use RNase‑Free ddH₂O for blanking. One absorbance unit at 260 nm corresponds to 40 μg/mL RNA.
Purity:
The A₂₆₀/A₂₈₀ ratio is used to assess nucleic acid purity. A ratio between 1.8 and 2.1 indicates high‑purity RNA. The A₂₆₀/A₂₈₀ ratio is dependent on pH and ionic strength; as pH increases, A₂₈₀ decreases while A₂₆₀ remains unchanged, leading to an increased ratio. Because water is typically acidic, it can lower the A₂₆₀/A₂₈₀ ratio. We recommend using a weakly alkaline buffer (e.g., TE, pH 8.0) as the diluent and blank control to obtain accurate, reproducible readings.
规格
别名
组织/细胞/细菌总RNA提取试剂盒 (DNaseI) (离心柱型) (含保存液)
英文别名
Total RNA Isolation Kit (Tissue/Cell/Bacteria)
规格或纯度
BioReagent,分子生物学级
英文名称
Tissue/Cell/Bacterial Total RNA Extraction Kit (DNase I & Preservation Solution)
应用
RNA Extraction
样品类型
组织, 细胞, 细菌
储存与运输
储存条件
2-8°C储存,室温
运输条件
低温运输
稳定性与储存
各组分在相应的储存条件下保质期15个月。
组分与储存
T1521439 | 组件名称 | 50T | 储存温度 |
T1521439A | 组织样本RNA保存液 | 100 mL | RT. |
T1521439B | Buffer TRL | 40 mL | RT. |
T1521439C | Buffer RW | 38 mL | RT. |
T1521439D | Buffer RW2 | 12 mL | RT. |
T1521439E | RNase-Free ddH₂O | 15 mL | RT. |
T1521439F | Proteinase K | 0.7 mL | RT. |
T1521439G | 吸附柱RA (含2mL收集管) | 1 EA×50 | RT. |
T1521439H | RNase-Free离心管 (1.5mL) | 1 EA×50 | RT. |
T1521439I | 1mL注射器 | 1 EA | RT. |
T1521439J | DNase I | 1 EA | 2-8℃. |
T1521439K | DNase I Buffer | 4 mL | 2-8℃. |
T1521439L | RNase-Free ddH₂O | 1 mL | 2-8℃. |
T1521439A, T1521439B, T1521439C, T1521439D, T1521439E, T1521439F, T1521439G, T1521439H, T1521439I：室温保存，15个月。
T1521439J, T1521439K, T1521439L：2-8℃保存，15个月。
加入β-巯基乙醇的Buffer TRL可在4℃放置1个月。
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
通过匹配包装上的批号来查找并下载产品的 COA，每批产品都进行了严格的验证，您可放心使用！
| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0636039 |
分析证书 | T1521439 |
技术文档和文章
溶液计算器
常见问题
什么是「分子生物学级」？
「分子生物学级」表示该产品经过加工和检测，适用于分子生物学和蛋白质组学。典型规格包括：低或不可检测的核酸酶/蛋白酶活性、低内毒素、受控的微生物限度，以及高批间重现性。
什么是「BioReagent」？
「BioReagent」表示该产品经过加工和检测，适用于生物和生化应用。规格可能包括内毒素限值、微生物控制、无菌性、低 DNase/RNase/蛋白酶活性，以及在相关情况下的生物活性验证。
本产品应如何储存？
请于2–8 °C条件下保存。需冷藏保存以维持规定的有效期。
本产品如何运输？
本产品采用湿冰冷藏运输。收货后请立即拆包，并按上述储存条件保存。
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每个订单都会随附分析证书（COA），您也可以从账户订单历史中下载。如需订购前的COA，请联系您的客户代表，或使用PDP上的"申请COA"按钮——我们将提供代表性批次的COA。
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